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DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 99: 95–102, 2012
doi: 10.3354/dao02460
Published June 13
Sponge white patch disease affecting the
Caribbean sponge Amphimedon compressa
H. Angermeier1, V. Glöckner1, J. R. Pawlik2, N. L. Lindquist3, U. Hentschel1,*
1
Julius-von-Sachs-Institute for Biological Sciences, University of Würzburg, 97082 Würzburg, Germany
Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington,
Wilmington, North Carolina 28403-5928, USA
3
Institute of Marine Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 28557, USA
2
ABSTRACT: We report on a novel sponge disease, hereafter termed ‘sponge white patch’ (SWP),
affecting the Caribbean sponge species Amphimedon compressa. SWP is characterized by distinctive white patches of variable size that are found irregularly on the branches of diseased
sponges. Nearly 20% of the population of A. compressa at Dry Rocks Reef, Florida, USA, showed
symptoms of SWP at the time of investigation (November 2007−July 2010). Approximately 21% of
the biomass of SWP individuals was bleached, as determined by volume displacement. Scanning
electron microscopy analysis showed severe degradation of bleached tissues. Transmission electron microscopy of the same tissues revealed the presence of a spongin-boring bacterial morphotype that had previously been implicated in sponge disease (Webster et al. 2002; Mar Ecol Prog
Ser 232:305−309). This particular morphotype was identified in 8 of 9 diseased A. compressa individuals investigated in this study. A close relative of the aforementioned disease-causing alphaproteobacterium was also isolated from bleached tissues of A. compressa. However, whether the
spongin-boring bacteria are true pathogens or merely opportunistic colonizers remains to be
investigated. Molecular fingerprinting by denaturing gradient gel electrophoresis (DGGE)
demonstrated a distinct shift from the microbiota of healthy A. compressa to a heterogeneous mixture of environmental bacteria, including several phylotypes previously implicated in sponge
stress or coral disease. Nevertheless, tissue transplantation experiments conducted in the field
failed to demonstrate infectivity from diseased to healthy sponges, leaving the cause of SWP in A.
compressa to be identified.
KEY WORDS: Porifera · Spongin-boring bacterium · Coral reef disease · Pathogenesis ·
Alphaproteobacterium
Resale or republication not permitted without written consent of the publisher
INTRODUCTION
Marine sponges (phylum Porifera) are among the
most ancient multicellular animals (metazoans), with
a fossil record dating back > 630 million years (Love
et al. 2009). Sponges are known for their interactions
with various types of microorganisms, including
viruses, bacteria, cyanobacteria, archaea, fungi, protozoa and single-celled algae (Hentschel et al. 2006,
Taylor et al. 2007, Webster & Taylor 2012). In general, these microorganisms serve as food particles
*Corresponding author.
Email: [email protected]
which are retained from seawater in the choanocyte
chambers, translocated into the mesohyl interior and
digested by phagocytosis. Many sponges, the socalled high microbial abundance (HMA) sponges or
bacteriosponges (Reiswig 1981, Hentschel et al.
2003), contain symbiotic microbial consortia within
their mesohyl matrix that may amount to nearly 40%
of their biomass (Vacelet 1975). To date, more than 17
different bacterial phyla and 12 candidate phyla
have been discovered from sponges, with the vast
majority of these microbes defying attempts at labo© Inter-Research 2012 · www.int-res.com
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96
Dis Aquat Org 99: 95–102, 2012
ratory cultivation (Webster et al. 2010, Schmitt et al.
2012, Simister et al. 2012).
As has been reported for corals and other marine
invertebrates (Webster 2007), sponges can also suffer
from diseases and bleaching. Disease development
typically starts with the appearance of discolored
patches, followed by tissue disintegration, leading to
the exposure of the sponge skeleton. Sponge diseases have thus far been reported from many geographic regions, including the Great Barrier Reef, the
Indo-Pacific, the Mediterranean and the Caribbean
(Harvell et al. 1999). Sponge diseases that have
already been reported include Aplysina red band
syndrome (ARBS) (Olson et al. 2006), Aplysina black
patch syndrome (Webster et al. 2008), sponge orange
band (SOB) disease of Xestospongia muta (Cowart et
al. 2006, López-Legentil et al. 2010, Angermeier et al.
2011), brown lesion necrosis or disease-like syndrome of Ianthella basta (Cervino et al. 2006, Luter et
al. 2010), spongin-boring necrosis of Rhopaloides
odorabile (Webster et al. 2002) and pustule disease of
Ircinia fasciculata and I. variabilis (Maldonado et al.
2010). Outbreaks of various sponge diseases have
indeed proven to be fatal on several occasions in different localities (Gaino et al. 1992, Vacelet et al. 1994,
Maldonado et al. 2010, Cebrian et al. 2011). Research
on sponge diseases has generally been hampered by
the fact that the death of any given sponge is often
hard to define and frequently goes unnoticed.
The marine sponge Amphimedon compressa,
commonly called the erect rope sponge, is found
throughout Florida, USA, the Bahamas and the
Caribbean (Zea et al. 2009). Individuals range up to
1 m in size and are characterized by a distinct red
coloration. A. compressa has osculae scattered all
over its surface, and its tissue is soft and flexible. A.
compressa belongs to the low microbial abundance
(LMA) category of sponges following the definition
of Hentschel et al. (2003), which implies that the
sponge mesohyl is visually devoid of microorganisms as judged by transmission electron microscopy
and that bacterial numbers and phylogenetic composition are similar to those of seawater. Several
secondary metabolites have been isolated from this
species, comprising fatty acids (Carballeira et al.
1998), sterols (Ballantine & Williams 1977) and the
alkaloid amphitoxin, which displays antibacterial
activities as well as pronounced antifeedant activities against diverse reef fish (Albrizio et al. 1995,
Thompson et al. 2010). In this study, we describe, to
our knowledge for the first time, a pathological condition of the rope sponge A. compressa, which we
name ‘sponge white patch’ (SWP) disease owing to
the irregular white patches that cover the body of
afflicted sponge individuals.
MATERIALS AND METHODS
Sponge collection
Samples of Amphimedon compressa (class Demospongiae, order Haplosclerida, family Niphatidae)
were collected by SCUBA diving at a depth of
4−30 m within the Florida Keys National Marine
Sanctuary at Dry Rocks Reef, Florida (25° 07’ 91’’ N,
80° 17’ 56’’ W) in November 2007 and at Conch Reef,
Florida along a line transect (24° 56’ 86’’ N, 80° 27’
23’’ W) in May and September 2009 and July 2010.
Altogether, 709 individuals of A. compressa,
grouped into either healthy (n = 538) or diseased
categories (n = 171), were investigated in this study.
The frequency of SWP-diseased individuals was
determined for Dry Rocks Reef by inspecting 80
randomly selected 1 m2 quadrats via SCUBA diving.
The sponge samples collected for further experimental work were transferred to the surface in seawater-containing Ziploc bags and were kept cool
until further processing within 1−2 h. The amount of
white and red tissue per SWP individual (n = 40
individuals) was determined by volume displacement using a seawater-filled graduated plastic cylinder. For each sponge, the healthy tissue was dissected immediately upon return to the marine
station from the diseased tissue prior to the analysis
using a scalpel, and the volume of seawater displaced by each of the 2 fractions was determined
independently.
Electron microscopy
Scanning electron microscopy (SEM) was performed on 0.5 cm3 sections of sponge tissue from
healthy as well as diseased (red and white) A. compressa specimens following established protocols by
Angermeier et al. (2011). This involved the excision
of sponge tissue, storage in 6.25% glutaraldehydephosphate-buffered solution, a 5× washing procedure with Soerensen-phosphate buffer (50 mM,
pH 7.4) for 5 min each and dehydration in an increasing acetone series starting with 30% for 15 min, followed by 50% for 20 min, 75% for 30 min, 90% for
45 min and 6× incubation at 100% for 30 min. The
samples were critical point dried in 100% acetone
under pressure, sputtered with gold-palladium and
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Angermeier et al.: Sponge white patch disease of A. compressa
stored in the dehydrator until examination with the
scanning electron microscope (Zeiss DSM 962).
Transmission electron microscopy (TEM) was performed on the same A. compressa individuals as used
for SEM following the procedure of Angermeier et al.
(2011). The prepared samples were sectioned with
an MT-7000 ultramicrotome (RMC) for examination
with the transmission electron microscope (Zeiss
EM10).
Denaturing gradient gel electrophoresis
For DNA extraction, 0.5 cm3 sponge samples were
preserved in 70% ethanol, air-dried and homogenized with a mortar and pestle in liquid nitrogen. The
DNA extraction and 16S rRNA gene amplification for
denaturing gradient gel electrophoresis (DGGE)
were performed as described in Angermeier et al.
(2011) with the primer pair 341f with GC-clamp and
907r (Muyzer et al. 1998), which resulted in a PCR
fragment of approximately 566 bp in size. The PCR
was conducted using a T3 thermocycler (Biometra)
with the following conditions: initial denaturation at
94°C for 2 min, 34 cycles of denaturation at 94°C for
1 min, primer annealing at 57°C for 30 s followed by
elongation at 72°C for 40 s and a final extension step
at 72°C for 5 min. Thirty-four PCR cycles were necessary to obtain a clearly visible DGGE pattern for this
LMA sponge. The size and quality of the obtained
PCR products were examined on 2% agarose gels
stained with ethidium bromide. DGGE was conducted on 10% (w/v) polyacrylamide gels with a
denaturing gradient of 0−100% (Muyzer et al. 1998).
Selected DGGE bands were excised with an ethanolsterilized scalpel under UV light and DNA was
eluted overnight with 25 µl H2O at 4°C. The excised
DGGE bands were re-amplified with the primer pair
341f and 907r using the following conditions: initial
denaturation at 94°C for 2 min followed by 29 cycles
of denaturation at 94°C for 1 min, annealing of the
primers at 60°C for 30 s and elongation at 72°C for
40 s. The final extension step was at 72°C for 5 min.
The obtained PCR products were purified, ligated
into the vector pGEM-T-Easy (Promega) and transformed into Escherichia coli Novablue cells. The
plasmid DNA was isolated by standard miniprep
procedures (Sambrook et al. 1989) and digested
with EcoRI to confirm the correct insert size by
agarose gel electrophoresis. Sequencing was conducted for one clone per DGGE band. Chimeras
were identified with the program Pintail (Ashelford
et al. 2005) by comparison against the 5 most closely
97
related 16S ribosomal ribonucleic acid (rRNA) gene
sequences from culturable bacteria and removed
from the data set. All 16S rRNA gene sequences
were deposited in GenBank (accession numbers
HQ659567−HQ659575).
Infection experiments
Sponge tissue transplantation experiments were
conducted along a 20 m transect at Conch Reef in
September 2009. Healthy or diseased tissues (n = 10
for each group) were attached to an equivalent number of healthy, randomly selected individuals of
Amphimedon compressa using cable ties. Infection
experiments of healthy A. compressa were performed with alphaproteobacterial strain HA007
(accession number JQ582943), which was previously
isolated from SWP A. compressa tissues and is closely
related to alphaproteobacterium strain NW4327
(Webster et al. 2002). Ten milliliters of both broth culture and a 1:10 dilution of broth in sterile seawater
(each n = 3) of this isolate were injected into healthy
A. compressa specimens. ZoBell medium without
bacterial inoculum served as negative control and
broth culture grown from 10 pooled A. compressa
white tissue patches served as positive control. The
recipient sponges were monitored daily by SCUBA
diving for up to 9 d, at which point the field work was
terminated.
RESULTS AND DISCUSSION
We report herein a disease phenomenon affecting
the sponge Amphimedon compressa from coral reefs
off Key Largo, Florida, USA. The disease was observed in winter 2007 at Dry Rocks Reef and in summer 2009 and 2010 also at Conch Reef, both within
the Florida Keys National Marine Sanctuary. We
named the disease ‘sponge white patch’ owing to the
large, distinct white patches that appear irregularly
on diseased sponge individuals (Fig. 1a−c). A transition zone between red and white tissues or inner and
outer regions was not observed; rather, the red and
white tissues were sharply separated from each other
(Fig. 1d).
To assess the impact of SWP disease on the population of Amphimedon compressa at Dry Rocks Reef, a
quantitative survey was performed by counting
healthy and diseased specimens. Of the 610 individuals of A. compressa surveyed, 82% (n = 503) was
visually healthy and 18% (n = 107) showed the spe-
Dis Aquat Org 99: 95–102, 2012
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98
Fig. 1. Amphimedon compressa. (a) Underwater and (b−d)
laboratory photographs of individuals with sponge white
patch disease collected at either (a,b) Conch or (c,d) Dry
Rocks Reef, Florida, USA
cific symptoms of disease. The population density of
A. compressa at this site was 7.6 ± 0.6 sponges m−2.
To quantify the impact of disease for a sponge individual, the amount of diseased tissue per sponge was
determined using the volume displacement method.
The mean tissue volume of diseased A. compressa
was 59.0 ± 7.4 ml (n = 40). On average, 78.8% (SD =
0.25, SE = 0.04) of the volume of a diseased individual
was healthy red tissue and 21.2% (SD = 0.25, SE =
0.04) was bleached.
To obtain more detailed insights into the disease
process, red tissues from healthy individuals of
Amphimedon compressa (n= 2) as well as red and
white SWP tissues from diseased ones (n = 3) were
inspected by SEM. The red tissue of healthy individuals (Fig. 2a,b) was indistinguishable from the red
tissue of SWP individuals (Fig. 2c,d). Round sponge
cells of 5−10 µm size were present in the red parts of
both healthy and diseased sponges. In stark contrast,
the white tissues of SWP individuals revealed obvious signs of destruction, including ample amounts of
detritus, exposure of spicules and the presence of
only few intact sponge cells (Fig. 2e,f).
TEM studies were conducted on healthy (n = 5) as
well as SWP (red and white tissue; n = 9) specimens
of Amphimedon compressa. Protective barriers of
densely compacted collagen, as were observed separating the diseased and healthy tissues in Ircinia
sponges (Maldonado et al. 2010), were not observed
in the present study. However, spongin-boring bacteria were found within the collagen fibers in 8 of 9
diseased individuals investigated and were seen to
produce an elaborate canal system (Fig. 3). The spongin-boring bacteria were approximately 0.5 × 1 µm
in size and appeared to be spherical or rod-shaped
(Fig. 3c). The images shown in Fig. 3 resemble
closely the only confirmed sponge pathogen so far
that has been identified as the spongin-boring
alphaproteobacterium NW4327 affecting Rhopaloides odorabile (Webster et al. 2002). Sponginboring bacteria were also observed in the spongin
matrix of healthy Axinella verrucosa (Steffens 2003).
Interestingly, a close relative of NW4327, termed
alphaproteobacterium strain HA007, was isolated
from A. compressa tissues (99.4% sequence identity
over 1375 bp; accession number JQ582943); however, infections of sponges with this isolate did not
result in a disease phenotype. Whether the sponginboring bacteria of A. compressa are true pathogens
in the sense of the Koch’s postulates or whether they
are merely secondary opportunistic colonizers that
degrade the exposed spongin matrix remains to be
determined in future studies. Nonetheless, the correlation between distinctive spongin-boring bacterial
morphotypes in bleached tissues of both R. odorabile
and A. compressa and the manifestation of a disease
condition in both sponge species is a relevant finding
which deserves further attention.
To elucidate the microbial community changes during disease progression, the microbial fingerprints of
the red versus white tissue of 3 SWP specimens of
Amphimedon compressa were assessed by DGGE
and compared with the microbial fingerprint of one
healthy individual (Fig. 4). Interestingly, the dominant DGGE band (#10) common to all red tissues was
absent in all white tissues. The bacterial clone
obtained from this DGGE band was most closely
99
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Angermeier et al.: Sponge white patch disease of A. compressa
Fig. 2. Amphimedon compressa. Scanning electron micrographs of a (a,b) healthy individual and the (c,d) red and (e,f) white
tissues of a sponge white patch (SWP) individual. Scale bars = (a,c,e) 50 µm and (b,d,f) 10 µm. Aq: aquiferous canal; D: detritus;
Sc: sponge cells; Sp: spicules
related (91% sequence identity) to a 16S rRNA gene
sequence derived from another sponge, Crambe
crambe (Table 1). Six more sequences from healthy A.
compressa sponges were obtained by DGGE that
belonged either to C. crambe-associated clone trans-
formant6 (GU799617) or to C. crambe-associated
clone transformant10 (GU799622) with similarly low
sequence identity values (data not shown). In contrast, the DGGE bands from the white A. compressa
tissues were most closely related to environmental
Dis Aquat Org 99: 95–102, 2012
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100
Ch
Sp
Ch
Ch
C
C
B
B
C
a
b
c
Fig. 3. Amphimedon compressa. Transmission electron micrographs of diseased tissues of a diseased individual. B: sponginboring bacteria; C: sponge collagen; Ch: channels; Sp: spicules. Scale bars = (a) 5 µm, (b) 1 µm and (c) 0.5 µm
H
#1
#2
#1
#3
1
2
34
#3
#2
8
9
5
6
7
10
Red
Red
Healthy
White
Diseased
Fig. 4. Amphimedon compressa. Denaturing gradient gel electrophoresis fingerprinting of a healthy (H) individual and of the
red (R) and white (W) tissues of diseased individuals (#1, #2 and #3). Arrows point towards the excised bands (1−10; see Table 1)
sequences (polluted sands, microbial flocs and engineered microfiltration systems), to a 16S rRNA gene
sequence derived from a heat stressed sponge, or
to phylotypes implicated in coral diseases (white
plague-like syndrome and black band disease).
Whether sponges act as reservoirs for coral-disease
associated bacteria, as has been proposed previously
(Ein-Gil et al. 2009, Negandhi et al. 2010), or whether
they are merely colonized by opportunistic bacteria
capable of utilizing decaying material remains to be
investigated. Indeed, many marine pathogens can be
found in the environment, so their recovery from
sponge tissues is not unexpected.
The shift from the normal host-associated microbiota towards a different mix of environmental phylotypes has been previously noted in diseases of
sponges (Webster et al. 2008, López-Legentil et al.
2010, Angermeier et al. 2011) and corals (i.e. Bourne
et al. 2008). Indeed, there has been a recent conceptual change, moving away from focusing on microbial pathogens as disease-causing agents to recognizing the relevance of intrinsic microbial community
shifts prior to or during disease progression (Webster
et al. 2008, Luter et al. 2010, Angermeier et al. 2011).
Even though there is experimental evidence for the
presence of bacteria implicated in sponge and coral
disease in SWP-afflicted Amphimedon compressa,
tissue transplantation trials failed to provoke a SWP
phenotype. Therefore, the cause of SWP in A. compressa cannot be unambigously identified. More
detailed experimental time series as well as longterm monitoring studies, taking into account also
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Angermeier et al.: Sponge white patch disease of A. compressa
101
Table 1. 16S rRNA gene sequence analysis of the DGGE bands shown in Fig. 4
Tissue
Diseased: white
Diseased: red
Band Accession
number
1
HQ659567
2
HQ659568
3
HQ659569
4
HQ659570
5
HQ659571
7
HQ659572
8
HQ659573
9
HQ659574
10
HQ659575
Closest sequence
match in GenBank
Similarity
(%)
Clone SGUS1003 associated
with aquarium-kept
Montastraea faveolata (FJ202771)
Clone 33aA1 associated with
Rhopaloeides odorabile
exposed to 33°C (EU183945)
Flavobacteriaceae isolate DGGE
gel band S12 obtained from
hydrocarbon and oil spill
polluted sand (EU375150)
Alphaproteobacterium clone 4-7-2
associated with white plague-like
syndrome affected Montastraea
annularis (AF544940)
Alphaproteobacterium clone RB_18f
associated with black band-diseased
Siderastrea siderea from the Bahamas
(EF123421)
Microfiltration system associated
bacterium clone MF-Oct-95 (HQ225056)
Montastrea faveolata associated
Clostridiaceae bacterium
clone MD3.13 (FJ425601)
Clone BBD-Dec07-1BB-2 associated
with the black band disease consortium
of Favia sp. (GQ215183)
Sponge Crambe crambe associated
clone transformant10 (GU799622)
Length
(bp)
Taxonomic
affiliation
99
583/586
Bacteria
99
439/441
Bacteria
89
474/531
Flavobacteriaceae
97
424/433
Alphaproteobacteria
98
553/560
Alphaproteobacteria
96
546/565
Bacteria
96
540/559
Clostridiaceae
99
561/564
Bacteria
91
538/589
Bacteria
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Submitted: August 2, 2011; Accepted: February 28, 2012
Proofs received from author(s): May 10, 2012